Enhanced transfer of DNA conjugates into cells has been achieved with adenovirus, a human DNA virus which readily infects epithelial cells (Horwitz, “Adenoviridae and Their Replication”, in Virology, Fields and Knipe, eds., Raven Press, NY (1990) pp. 1679–1740).
Although adenovirus-mediated gene therapy represents an improved method of DNA transfer into cells, a potential limitation of this approach is that adenovirus replication results in disruption of the host cell. In addition, adenovirus also possesses oncogenic properties including the ability of one of its proteins to bind to tumor suppressor gene products. The use of so-called replication defective strains of adenovirus (which typically possess E1A and/or E1B deletions that render the virus unable to replicate in host cells) is in principle more suitable for in vivo therapy; however, the potential of co-infection of epithelial cells with wild-type strains of virus resulting in transactivation of the recombinant virus may represent a significant safety concern for in vivo applications.
Another undesirable aspect of using intact or replication-competent adenovirus as a gene transfer means is that it is an oncogenic virus whose gene products are known to interfere with the function of host cell tumor suppressor proteins as well as immune recognition molecules, such as the major histocompatibility complex (MHC). In addition, pre-existing circulating antibodies to adenovirus may significantly reduce the efficiency of in vivo gene delivery. Lastly, only a foreign gene of 6 kilobases (kb) or less can be incorporated into the intact adenovirus genome for gene transfer experiments, whereas DNA segments of greater than 12 kb can be transferred using the methods of this invention.
In order to make Ad vectors more replication-incompetent, some investigators have attempted to construct recombinant Ad-derived vectors which have nearly all of their genome deleted, except for portions known to be required for packaging of virus particles. For example, helper-dependent vectors lacking all viral ORFs but including essential cis elements (the inverted terminal repeats—ITRs—and the contiguous packaging sequence) have been constructed, but the virions package less efficiently than the helper and package as multimers part of the time, which suggests that the virus may “want” to package a fuller DNA complement (see, e.g., Fisher, et al., Virology 217: 11–22, 1996). Mitani et al. (Proc. Natl. Acad. Sci. USA 92: 3854–3858, 1995) also describe a helper-dependent Ad vector that was apparently not completely replication-defective.
Amalfitano, et al. (Proc. Natl. Acad. Sci. USA 93: 3352–3356, 1996) describe the construction of Ad packaging cell lines that support the growth of E1- and polymerase-deleted Ad vectors, in an effort to block the replication of Ad vectors in vivo. Similarly, Armentano, et al. (Hum. Gene Ther. 6: 1343–53, 1995) describes Ad vectors with most—but not all—of the E4 sequence deleted therefrom. However, since such a small amount of genetic material is deleted from the vectors, their ability to transport therapeutic sequences is rather limited. Published International App. No. WO96/14061 describes efforts to construct packaging cell lines containing nucleotide sequences encoding E1 and ORF6 of E4.
In addition to being able to incorporate large amounts of DNA into a vector, the ability to target the vectors to specific cell types will result in more efficient administration of desired therapeutics. Such targeting of adenovirus in a relatively simple system is one of the advantages of the current invention.
Thus, there is a need in the art to obtain Adenovirus vectors 1) capable of incorporating large segments of foreign DNA and capable of being targeted to specific cells, as well as to obtain cell lines which can package such adenovirus-gene deficient vector or targeted vectors. These needs, as well as others, are met by the invention.